Hospital-acquired pneumonia (HAP) and sepsis are leading causes of morbidity and mortality in critically ill patients, especially in hospital intensive care units. These acute conditions are caused by infection with a range of Gram-positive and Gram-negative bacteria commonly found in the hospital environment. Treatment of patients with suspected HAP or sepsis is often initiated based on presumptive evidence. The presence and identity of the pathogen are generally not established until culture results are available (usually days after samples are obtained), followed by a further delay for completion of antibiotic susceptibility testing. Therefore broad-spectrum antibiotics are commonly administered by default. However, an increasing proportion of bacterial pathogens exhibit drug resistance to these antibiotics, and conventional testing may miss clinically relevant resistances, resulting in treatment failures and increased patient mortality. Thus, determining as early as possible both the identity of bacteria causing HAP or sepsis and their drug resistance profiles is critical to making treatment decisions, and modifying or de-escalating antibiotic usage. The aim of the proposed project is to develop a molecular test for concurrent detection and identification of microorganisms and genetic determinants of antibiotic resistance, including resistance to [unreadable]-lactams and fluoroquinolones in Gram-negative bacteria, and to methicillin and vancomycin in Gram-positive bacteria. The diagnostic test will be based on a reverse line blot assay developed by investigators, which enables detection and identification of a wide range of common bacterial pathogens. In contrast with the 2-4 day delay required for culture-based methods, it will offer same-day turnaround by obtaining genetic information directly from clinical samples without need for bacterial growth. In Phase I, the assay will be developed based on respiratory and blood samples from patients with suspected HAP or sepsis obtained from intensive care units at two hospital sites. Results of the reverse line blot method will be compared with those of conventional culture-based ID and antibiotic susceptibility testing. In Phase II, a commercial version of the assay will be completed and its accuracy in predicting antibiotic resistant phenotypes, and thereby potentially improving treatment decisions and healthcare outcomes for affected patients in hospital intensive care units, will be evaluated in a prospective study. Overall, this novel molecular diagnostic assay will provide a powerful tool in efforts to improve treatment and limit the emergence and spread of drug resistant bacteria by appropriate antimicrobial use and infection control measures. PUBLIC HEALTH RELEVANCE: Hospital-acquired pneumonia (HAP) and sepsis resulting from bacterial infection are leading causes of morbidity and mortality in hospital intensive care units, and the emergence of antibiotic resistance in many bacteria is a major threat to effective treatment. This application will result in the development of a rapid test for simultaneous identification of bacterial species and drug resistance genes in bacteria causing these conditions. Earlier detection of the bacteria and their antibiotic resistances than is now possible will enable clinicians to select more effective antibiotic therapy, improving the survival rate for affected patients and deterring the spread of drug resistant bacteria.